摘要
应用一步法逆转录—聚合酶链式反应 (RT_PCR)技术对罗曼蛋鸡γ_干扰素基因 (CHIFN_γ)进行了扩增。试验结果表明 ,在植物血凝素 (PHA) 5 0 0 μg/mL、细胞浓度 2 .5× 1 0 6/mL、诱导 1 0h,能从外周血淋巴细胞和脾淋巴细胞的总RNA中扩增出特异性片段。将RT_PCR产物克隆到PGEM_Teasy载体 ,经过限制性酶切分析、测序证实克隆到的CHIFN_γ基因正确 ,命名为PGEM_CHIFN_γ。序列分析表明 ,CHIFN_γ基因与已发表的鸡IFN_γ基因的同源性为 95 .6%～ 1 0 0 % ,氨基酸水平同源性为 92 .8%～ 1 0 0 % ,与鸭IFN_γ核苷酸同源性为 67.5 %。将CHIFN_γ基因克隆到原核表达载体pGEX_6P_1的GST基因的下游 ,经IPTG诱导后表达出了 43kD的融合蛋白 ,超声波裂解后发现融合蛋白主要以包涵体的形式存在 ,裂解上清无抑制病毒活性 ;然而将该基因克隆到真核表达载体pcDNA3 .1中 ,通过脂质体转染COS_1细胞 ,用鼠抗CHIFN_γ高免血清进行间接免疫荧光试验 (IFA) ,结果表明在COS_1细胞的细胞膜和胞浆内均有CHIFN_γ表达 ,且细胞病变抑制试验证实 ,转染后细胞培养上清有干扰素活性 ,在鸡胚成纤维细胞 (CEF)上抑制VSV病毒的效价可达到 1 0 2 4U/ml。这些结果表明真核表达系统对CHIFN_γ基因表达产物的活性有重要影响。
Chicken interferon_gamma (CHIFN_γ) gene was amplified by reverse transcription polymerase chain reaction (RT_PCR) from total RNA of chicken lymphocytes stimulated with Phytohemagglutinin (PHA). The products of RT_PCR were cloned into pGEM_T easy vector。All of the recombinant plasmid DNA were indentificated with restriction enzyne digestion,and postive clones were named as CHIFN_γ1. The sequence of the gene showed 95.6 %～100 % homologous compared with other published CHIFN_γ genes, but67.5 % homologous compared with duck interferon_γ. The fragment of CHIFN_γwas subcloned into pGEX_6P_1. Unsoluble 43KD GST_CHIFN_γ fusion protein was expressed after induced by IPTG, however the supernatant of bacterial sonicates did not show any antiviral activitie. It is interesting that antiviral activity was observed when COS_1 cells was tansfected with pcDNA_CHIFN_γ, which was constructed with CHIFN_γ gene and mammalian expression vector pcDNA3.1. The titer of antiviral activity in the supernatant of COS_1 cells transfected with pcDNA_CHIFN_γ was up to 1 024 U per milliliter. The rCHIFN_γ were detected in the cytoplasms and cytomembranes of COS_1 cells by immunofluorescence assay.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2003年第4期249-253,共5页
Chinese Journal of Preventive Veterinary Medicine
关键词
鸡
Γ-干扰素
淋巴细胞
克隆
表达
chicken
interferon_gamma
lymphocyte
clone
expression
